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- PDB-8uc1: Cryo-EM structure of dolphin Prestin in low Cl buffer -

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Basic information

Entry
Database: PDB / ID: 8uc1
TitleCryo-EM structure of dolphin Prestin in low Cl buffer
ComponentsPrestin
KeywordsMEMBRANE PROTEIN / Solute Carrier / Electromotility / Voltage Sensitive / Mechanosensitive
Function / homology
Function and homology information


cochlear outer hair cell electromotile response / secondary active sulfate transmembrane transporter activity / sensory perception of sound / regulation of cell shape / plasma membrane
Similarity search - Function
Sulphate anion transporter, conserved site / SLC26A transporters signature. / SLC26A/SulP transporter / SLC26A/SulP transporter domain / Sulfate permease family / STAS domain / STAS domain profile. / STAS domain / STAS domain superfamily
Similarity search - Domain/homology
Biological speciesTursiops truncatus (common bottlenose dolphin)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsHaller, P. / Bavi, N. / Perozo, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)5R01DC019833-03 United States
CitationJournal: Elife / Year: 2023
Title: Folding of prestin's anion-binding site and the mechanism of outer hair cell electromotility.
Authors: Xiaoxuan Lin / Patrick R Haller / Navid Bavi / Nabil Faruk / Eduardo Perozo / Tobin R Sosnick /
Abstract: Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs ...Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin's voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl anion at a conserved binding site formed by the amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin's mechanical expansion. We observe helix fraying at prestin's anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin's fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and help define prestin's unique voltage-sensing mechanism and electromotility.
History
DepositionSep 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Prestin
B: Prestin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)162,0184
Polymers161,9482
Non-polymers712
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Prestin / / Solute carrier family 26 member 5


Mass: 80973.750 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tursiops truncatus (common bottlenose dolphin)
Gene: SLC26A5 / Production host: Homo sapiens (human) / References: UniProt: D7PC76
#2: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dolphin Prestin solubilized in GDN in low Cl buffer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Tursiops truncatus (common bottlenose dolphin)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Details: 190 mM HEPES, 95 mM Tris-base, 1mM NaCl, 3mM DTT, 1mM EDTA, 0.02 % GDN
Buffer component
IDConc.NameFormulaBuffer-ID
1190 mMHEPESC8H18N2O4S1
21 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: 3.5s Blot time, Blot force 1

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 700 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.2 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameCategory
2EPUimage acquisition
7Cootmodel fitting
8PHENIXmodel fitting
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 170000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7S8X

7s8x


Accession code: 7S8X / Source name: PDB / Type: experimental model

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